Start codon disruption with CRISPR/Cas9 prevents murine Fuchs' endothelial corneal dystrophy.

A missense mutation of collagen type VIII alpha 2 chain (COL8A2) gene leads to early-onset Fuchs' endothelial corneal dystrophy (FECD), which progressively impairs vision through the loss of corneal endothelial cells. We demonstrate that CRISPR/Cas9-based postnatal gene editing achieves structural and functional rescue in a mouse model of FECD. A single intraocular injection of an adenovirus encoding both the Cas9 gene and guide RNA (Ad-Cas9-Col8a2gRNA) efficiently knocked down mutant COL8A2 expression in corneal endothelial cells, prevented endothelial cell loss, and rescued corneal endothelium pumping function in adult Col8a2 mutant mice. There were no adverse sequelae on histology or electroretinography. Col8a2 start codon disruption represents a non-surgical strategy to prevent vision loss in early-onset FECD. As this demonstrates the ability of Ad-Cas9-gRNA to restore the phenotype in adult post-mitotic cells, this method may be widely applicable to adult-onset diseases, even in tissues affected with disorders of non-reproducing cells.

Does squalene alter the antioxidant potential of astaxanthin and fucoxanthinol? In vitro evidence in RAW 264.7 cells, a murine macrophage.

Astaxanthin (Ax) and fucoxanthin/fucoxanthinol (FuOH) are marine xanthophylls exhibiting anti-oxidant effects. Squalene (SQ) is a triterpenoid and is a precursor of sterols. This study aimed to determine if SQ can improve the effect of Ax/FuOH on lipid peroxidation. RAW 264.7 cells were treated with different concentrations of Ax, FuOH and SQ and corresponding rate of cell survival was noted. In addition,combination groups - Ax + SQ and FuOH + SQ- were also run. Cells treated with Ax, FuOH, SQ, Ax + SQ and FuOH + SQ were stimulated with lipopolysaccharide and lipid hydroperoxides were estimated. Results showed that 5 µM Ax, 2 µM FuOH and 10 µM SQ supported cell survival. In presence of SQ, cell viability improved for higher concentrations of FuOH (5, 10 µM). Lipid hydroperoxides were supressed by Ax, FuOH, Ax + SQ and FUOH +SQ and were significantly lower in Ax + SQ, indicating the synergistic effect of Ax and SQ. To conclude, combination of Ax with SQ enhances its ability to supress lipid peroxidation while with FuOH, SQ attenuates the toxic effect at higher doses. Moreover, this is the first time that the combined effect of SQ and carotenoids has been studied and reported.

Combined effect of astaxanthin and squalene on oxidative stress in vivo.

Obesity and diabetes, risk factors for metabolic syndrome, are characterized by oxidative stress and inflammatory responses. Marine biofunctionals, astaxanthin (Ax) and squalene (SQ), were evaluated for their combined effect. Groups of male KK-A (y) mice were fed high fat/sucrose diet for 4 weeks, supplemented with either 0.1 %Ax, 2 %SQ or 0.1 %Ax + 2 %SQ. In comparison to control, Sod was elevated in only Ax + SQ. However, Gpx was highest in Ax + SQ, indicating the combined antioxidant effect of Ax and SQ. This was supported by elevated mRNA expression of Sod1 and Gpx1. Except adiponectin (elevated in Ax and Ax + SQ), expression of other inflammatory markers was not altered. Blood glucose levels were decreased in SQ and Ax + SQ while liver triglycerides decreased in SQ group. This is the first in vivo study demonstrating combined effects of Ax and SQ resulting in antioxidant effects and modulation of glucose/triglyceride levels. This study highlights the benefit of utilizing Ax and SQ together for management of obesity/diabetes.

Fucoxanthin restrains oxidative stress induced by retinol deficiency through modulation of Na(+)K(+)-ATPase [corrected] and antioxidant enzyme activities in rats.

BACKGROUND: Retinol deficiency is a major public health problem world wide, affecting children and women, in particular. It causes a variety of disorders in the body affecting various cellular functions. AIM OF THE STUDY: To study the effect of fucoxanthin (FUCO), a non-provitamin-A carotenoid in comparison with retinol (ROH) on changes in antioxidant molecules, lipid peroxidation and membrane bound enzymes in tissue and microsomes, induced by ROH deficiency in rats. METHODS: After induction of ROH deficiency by feeding a diet devoid of ROH for 8 weeks, rats were divided into two groups (n = 20/group) and administered orally a dose of either FUCO (0.83 micromol) or ROH (0.87 micromol). A group of ROH deficient rats (n = 5) and rats (n = 5) fed with ROH sufficient diet was considered as baseline and control groups respectively. Over a period of 8 h, activity of catalase (CAT), glutathione transferase (GST), level of lipid peroxidation (LPx), fatty acids in plasma, liver and liver microsomes and activity of Na(+)K(+)-ATPase in liver microsomes were evaluated. RESULTS: ROH restriction increased LPx (P < 0.05) in liver (~19%) and plasma (~34%) while the activities of CAT (90 +/- 1%) and GST (17 +/- 4%) decreased compared to control. Significant elevation (91%) was observed for Na(+)K(+)-ATPase activity in liver microsomes of ROH deficient when compared to control group and levels were lowered on administration of ROH (37-69%) and FUCO (51-57%), towards control over a period of 8 h. ROH and FUCO suppressed (P < 0.05) the LPx level (%) in plasma (34-62, 7-85), liver homogenate (9-71, 24-72) and liver microsomes (83-92, 61-87), while the activities of CAT in plasma (89-97%, 91-95%) and liver microsomes (84-93%, 85-93%) and GST in liver homogenate (43-53%, 44-51%) and liver microsomes (36-52%, 22-51%) were increased (P < 0.05) compared to ROH deficient group. CONCLUSIONS: Results show that FUCO, a non-provitamin-A carotenoid protects cell membrane by modulating Na(+)K(+)-ATPase (51-57% lowering) and the activities of CAT and GST at the tissue and microsomal level which are affected by ROH deficiency. This may be due to its antioxidant nature. These in turn reduce LPx caused by ROH deficiency.